JP5545683B2 - Immunochromatographic test equipment - Google Patents

Description

  The present invention relates to an immunochromatographic test device.

  An immunochromatographic method combining a chromatographic method using a capillary phenomenon and an immunological method is widely used as a method capable of analyzing an analyte in a sample in a simple and short time.

  FIG. 1 shows a conventional general immunochromatographic test device. On the back sheet 5 in the order of the development pad 3, the conjugate pad 2, the reaction membrane 1, and the absorption pad 4 from the upstream (left end in the figure) to the downstream (right end in the figure) in the development direction. Is arranged. The conjugate pad is impregnated with a first antibody that specifically binds to the analyte and is labeled with colloidal gold particles or the like. The reaction membrane has a test line 6 on which a second antibody that specifically captures an analyte at a site different from the first antibody is immobilized.

  Next, analysis of the analysis substance using this immunochromatographic test device will be described. When the test solution is supplied to the development pad 3, it moves to the conjugate pad 2 by capillary action. Then, the labeled first antibody impregnated in the conjugate pad is eluted in the test solution. The test solution in which the labeled first antibody is dissolved sequentially develops to the test line 6 on the reaction membrane 1 by capillary action. Here, if the sample contains an analyte, the complex of the labeled first antibody and the analyte formed during the development is captured by the second antibody immobilized on the test line. As a result, the analyte in the sample can be analyzed by determining the presence or absence or degree of signal generated by the captured labeling substance.

However, in the conventional configuration, the detection sensitivity decreases due to some residue of the labeling substance on the conjugate pad, the background of the reaction film increases due to the re-supply of the residual labeling substance to the reaction film due to evaporation of the developing solution, etc. This problem occurred frequently and became an obstacle to result determination.
In order to avoid such a problem caused by the remaining of the labeling substance, an immunochromatographic test device in which the conjugate pad and the reaction membrane are arranged so as not to contact each other is considered useful. For example, Patent Document 1 provides an immunochromatographic analyzer having a reaction port in which a developing solution containing an analyte is held between a conjugate pad and an immunochromatographic developing membrane. Further, Patent Document 2 provides a test tool in which a developing member is provided between a label holding member and a chromatographic membrane carrier.

JP 2009-229343 A JP 2006-194687 A

However, in the analyzer of Patent Document 1 for the purpose of improving the detection sensitivity, the developing solution eluting the labeling substance is once held in the reaction port in order to sufficiently react the analyte and the labeling substance. It is considered that the labeling substance diffuses throughout the developing solution at the reaction port. Therefore, there is a concern that a part of the labeling substance diffused at the end of the development stagnates on the reaction film, and the background of the entire reaction film including the determination part increases. Furthermore, since it is necessary to arrange each pad individually in a casing having a complicated structure, the manufacturing process is also complicated.
Further, in the test device of Patent Document 2, for the purpose of increasing the elution rate of the labeling substance, the development member is arranged with a gap of preferably 5 mm or more between the label holding member and the chromatographic membrane carrier. However, since the flow of the developing solution is disturbed in the developing member, the labeling substance is diffused, and similarly to Patent Document 1, the background of the entire membrane is increased.

  Accordingly, an object of the present invention is to provide an immunochromatographic test device that suppresses the increase in background and improves the visibility of the test line by preventing the labeling substance from remaining on the pad and diffusion in the developing solution. It is in.

  As a result of intensive studies to solve the above problems, the present inventors have improved the elution of the labeling substance on the conjugate pad by providing a space between the conjugate pad and the reaction membrane, and the residual labeling substance The present inventors have found that the background can be expanded to the test line on the reaction film without diffusing the labeling substance while suppressing an increase in the background due to the resupply of the liquid, and the present invention has been completed.

That is, the present invention
(1) a conjugate pad holding a labeling substance;
(2) a reaction membrane disposed at a distance from the conjugate pad and having a site where a substance specific to the analyte is immobilized;
(3) a deployment pad arranged to generate capillary action with both the conjugate pad and the reaction membrane, wherein a space is formed between the conjugate pad and the reaction membrane; The test pad for immunochromatography, wherein the development pad is disposed on
About.

  By using the immunochromatographic test device of the present invention, the elution of the labeling substance is improved, and the labeling substance complex can be developed to the test line without diffusing, thereby suppressing an increase in background. it can. As a result, the visibility of the test line is improved, and a clear and reliable determination result can be obtained.

FIG. 2 is a schematic cross-sectional view of a conventional general immunochromatographic test device (provided that a sample addition pad is omitted) used in Comparative Example 1; It is a typical sectional view (same as the above) of the immunochromatographic test device used in Comparative Example 2. FIG. 6 is a schematic cross-sectional view (same as above) of an immunochromatographic test device used in Comparative Example 3. 1 is a schematic cross-sectional view of one embodiment of an immunochromatographic test device of the present invention. It is sectional drawing of the one aspect | mode of the immunochromatographic test device of this invention used for the Example.

  “Analysis” in the present specification includes both “measurement” for quantitatively or semi-quantitatively determining the amount of an analyte and “detection” for determining the presence or absence of the analyte.

  The immunochromatographic test device of the present invention is an improved invention of a conventionally known immunochromatographic test device, and is a conventionally known immunochromatographic device except that a space is provided between the conjugate pad and the reaction membrane. It can consist of the same composition as a testing device.

  Hereinafter, the immunochromatographic test device of the present invention will be described with reference to the accompanying drawings. For example, one embodiment of the immunochromatographic test device of the present invention shown in FIG. 4 is a development pad 3 for introducing a sample onto a backsheet 5 having an adhesive layer on the surface, and a label that specifically binds to an analyte. A conjugate pad 2 that holds the first substance, a reaction film 1, and an absorption pad 4 are provided.

  The conjugate pad and the reaction membrane are not in direct contact with each other, and are connected to each other so as to cause capillary action through the development pad. Here, a space is formed between the conjugate pad and the reaction membrane by arranging the development pad so as not to contact the back sheet on the reaction membrane side. If the development pad is in contact with a non-water-absorbing member such as a back sheet on the reaction membrane side, the migration speed at the contact area changes and the development pattern is likely to be disturbed. Preferably, the deployment pad does not directly contact other members. The distance between the conjugate pad and the reaction membrane may be any distance that does not allow each member to contact, but it is easy to prevent liquid leakage to the side surface and the spread pad from contacting the back sheet. Preferably, it is greater than 0 mm and within 2 mm, more preferably 1 mm to 2 mm.

  On the reaction membrane, there is a determination unit composed of a test line on which a second substance that specifically captures an analyte is immobilized and a control line on which a third substance that captures a labeled first substance is immobilized. The reaction membrane, the development pad, and the absorption pad are in a liquid receiving relationship, and the reaction membrane overlaps the development pad on the upstream end side and the absorption pad on the downstream end side.

  In the immunochromatographic test device of the present invention, by using an adhesive back sheet, the arrangement position of each member can be fixed and the connection between the members can be strengthened. As the material of the sheet, various materials such as paper or plastic can be used. In addition, the adhesive can be selected from those that do not interfere with all the reagents used in carrying out the assay.

  In the immunochromatographic test device of the present invention, a sample addition pad can be provided if desired. The shape and material of the sample addition pad are not particularly limited as long as the introduced sample solution can be temporarily absorbed and held and then supplied to the next pad. Although it is possible to add a sample directly to the development pad, it is preferable to provide a sample addition pad because it can easily have a function of filtering insoluble matters in the sample. Although the sample addition pad may be arranged so as to be in direct contact with the conjugate pad, it may be arranged so as to be connected via the development pad as shown in FIG. The sample solution includes not only a test sample containing an analysis substance or a processed product thereof (for example, a diluted product) but also various reagents (for example, signal generating components, antigens, antibodies) to the test sample or the processed product thereof. Etc.) are also included.

  The material of the conjugate pad used in the present invention is not particularly limited as long as it can move the analyte and the first labeled substance by capillary action. However, the material has both the liquid holding ability and the liquid releasing ability. Examples thereof include glass fiber, general filter paper, sponge, and cotton. More specifically, for example, a fiber sheet made of glass fiber, polyvinyl alcohol (PVA) fiber, cellulose fiber, rayon fiber or the like, or a mixture of the fibers (for example, a combination of glass fiber and PVA fiber, cellulose and glass) A fiber sheet made of a combination of glass fiber and PVA fiber, a fiber sheet made of a combination of cellulose and glass, and a fiber sheet made of rayon.

  The length and width of the conjugate pad are not particularly limited as long as a predetermined amount of labeling substance can be retained, and can be determined as appropriate. In addition, the length in this specification means the distance along the major axis direction of the immunochromatographic test device, and the width means the distance along the minor axis direction of the immunochromatographic test device.

The first substance retained on the conjugate pad is selected from substances that react specifically with the analyte, and examples include antigens and antibodies. The antibody may be used as an F (ab ′) ₂ , Fab ′, or Fab fragment by enzymatic treatment or chemical treatment. The labeling substance is not particularly limited as long as a signal that can be visually detected is obtained, and the one labeled with the first substance can be moved by capillary action. In addition to metal colloids such as platinum colloids or silver colloids, latex colored particles of organic polymers such as polystyrene and styrene-butadiene copolymers, liposomes and microcapsules containing dyes, and the like can be mentioned. The binding between the first substance and the labeling substance can be performed by a conventional method.

  As the material of the development pad used in the present invention, it is preferable to use the same material as that of the conjugate pad because the development pattern is not easily disturbed. The length of the development pad can be appropriately determined according to the member to be connected and the arrangement position thereof, and the width thereof is the same as that of the conjugate pad.

As a material for the reaction membrane in the present invention, for example, a porous membrane such as nitrocellulose, cellulose, nylon, polyvinylidene difluoride (PVDF), glass fiber or the like can be used.
The reaction film may include only one test line or two or more test lines. The second substance immobilized on the test line is selected from substances that specifically bind to the analysis substance, and examples thereof include antigens and antibodies. The antibody may be used as an F (ab ′) ₂ , Fab ′, or Fab fragment by enzymatic treatment or chemical treatment.
Further, the reaction membrane is provided with a control line on the downstream side of the test line, if desired. The third substance immobilized on the control line is selected from substances that react with the labeled first substance, and can be appropriately determined from a combination of antigen-antibody or ligand-receptor.

  Examples of the material of the absorbent pad in the present invention include water-absorbing materials such as cellulose filter paper, nonwoven fabric, and cloth. The sample development speed after the developing solution reaches the absorption pad differs depending on the material, size, etc., and can be determined as appropriate according to the analysis conditions.

  Examples of substances to be analyzed by the immunochromatographic test device of the present invention include proteins, polysaccharides, allergens, pathogens such as bacteria and viruses, but are not limited thereto.

The behavior of the introduced sample solution until it reaches the reaction membrane in the immunochromatographic test device of the present invention will be described.
When the sample is introduced into the development pad, the sample moves to the conjugate pad through the development pad by capillary action, and the labeled first substance impregnated in the conjugate pad is eluted into the sample solution. The sample solution eluting the labeled first substance quickly flows into a narrow space formed by the development pad and the back sheet by surface tension. Thereby, the labeled first substance can be eluted from the conjugate pad without delay. Thereafter, the sample solution that has flowed into the space is sequentially supplied to the reaction membrane through the development pad. Since the development pad is not in contact with the back sheet or the like, it is possible to minimize the disturbance of the development pattern. Just before the end of the development, a sample solution containing almost no labeled first substance is supplied to the reaction membrane.

  Thus, in the immunochromatographic test device of the present invention, the elution of the labeling substance can be enhanced by the action of the space arranged between the conjugate pad and the reaction membrane, and the conventional test device shown in FIG. As described above, it is possible to suppress a decrease in detection sensitivity due to a part of the labeling substance remaining on the conjugate pad and an increase in the background of the reaction film due to resupply of the residual labeling substance to the reaction film due to evaporation of the developing solution. Is possible. In addition, because of the function of the space, the flow of the developing solution eluting the labeling substance can be supplied to the reaction membrane in order without disturbing the flow, so that in the developing solution as in the conventional test device shown in FIG. The labeling substance does not diffuse, and it is possible to suppress an increase in background due to the labeling substance stagnating on the reaction film at the end of the development.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

Example 1: Production of immunochromatographic test device
In accordance with the following method, a labeling reagent and a control reagent were prepared, and then a conjugate pad and a reaction membrane were prepared, and further, an immunochromatographic test device was prepared using them.
(1) Preparation of labeling reagent (1-1) Preparation of streptavidin-immobilized gold colloid 10 mL of gold colloid solution (particle size) prepared with 5 mmol / L phosphate buffer pH 7.5 so that the absorbance at 520 nm is 10. Streptavidin (Wako Pure Chemical Industries, Ltd.) was mixed with 40 nm, Tanaka Precious Metals) so that the final concentration at the time of mixing was 0.1 mg / mL.
After the mixture was stirred for 1 hour, a polyethylene glycol (Mw. 20000, Wako Pure Chemical Industries) solution was added to a final concentration of 0.5%, and the mixture was further stirred for 1 hour. Subsequently, BSA (SIGMA) solution was added to a final concentration of 1%, and the mixture was stirred for 1 hour for blocking. This solution was centrifuged at 10000 × g for 10 minutes at 4 ° C. to remove the supernatant, and a dispersion buffer (5 mmol / L phosphate buffer pH 7.5 containing 1% BSA and 0.05% polyethylene glycol) was added. In addition, it was redispersed. Centrifugation was again performed at 4 ° C., 10,000 × g for 10 minutes, and the supernatant was removed and redispersed in a dispersion buffer to prepare a streptavidin-immobilized gold colloid solution.

(1-2) Preparation of Biotinylated Antibody (Capture Reagent) Anti-Mycoplasma pneumoniae 2 mg / mL F (ab ′) ₂ derived from mouse monoclonal antibody (MCM12) and SH group reducing agent (TCEP: PIERCE) at a final concentration of 0 After adding to 4 mmol / L to make Fab ′, maleimidated biotin reagent EZ-Link Maleimide-PEG2-Biotin (spacer length 29.1 mm, PIERCE) was adjusted to a final concentration of 0.4 mmol / L. And bound. This reaction product was buffer-exchanged to 50 mmol / L phosphate buffer pH 7.5 using an ultrafiltration membrane having a fractional molecular weight of 10,000 to prepare a biotinylated antibody.

(1-3) Preparation of Labeling Reagent The biotinylated antibody obtained in (1-2) was mixed with 10 mL of the streptavidin-immobilized gold colloid solution (OD _{520 nm} = 9) obtained in (1-1). Add to a concentration of 0.14 mg / mL, stir at 25 ° C. for 1 hour, leave at 4 ° C. for 20 hours, centrifuge at 4 ° C., 10,000 × g for 10 minutes to remove the supernatant and disperse The working buffer was added and redispersed. Centrifugation was again performed at 4 ° C., 10000 × g for 10 minutes, and the supernatant was removed and re-dispersed in a dispersion buffer to obtain a labeling reagent.

(2) Preparation of Control Reagent A 12 mg / mL BSA (SIGMA) biotinylated reagent EZ-Link NHS-PEG12-Biotin (spacer length 56 mm, PIERCE) that reacts with amino groups is adjusted to a final concentration of 3.2 mmol / L. Then, the solution was exchanged with a 50 mmol / L phosphate buffer (pH 7.5) using an ultrafiltration membrane with a molecular weight cut off of 50,000, and used as a control reagent.

(3) Preparation of immunochromatographic test device (3-1) Preparation of conjugate pad The labeling reagent prepared in (1) was prepared with a dispersion buffer so that OD _{520 nm} = 4.5. This was impregnated with 10 μL of a 5 mm × 6 mm glass fiber pad (Millipore) and dried overnight at room temperature to prepare a conjugate pad.

(3-2) Preparation of reaction membrane An anti-Mycoplasma pneumoniae mouse monoclonal antibody (MCM19) solution prepared to be 2 mg / mL as a test line on a 30 mm × 6 mm nitrocellulose membrane (Millipore), 2 mg / ml as a control line The control reagent (2) prepared to be mL was applied in a line shape having a width of about 1 mm using a coating machine (BioDot) and dried to prepare a reaction film. The control line was provided at a position 9 mm from the downstream end of the nitrocellulose membrane, and the test line was provided at a position 14 mm from the downstream end of the nitrocellulose membrane.

(3-3) Assembly of immunochromatographic test device Each member was affixed on a 69 mm x 6 mm back sheet (Nichiei Kako) as follows to prepare an immunochromatographic test device. The reaction film prepared in (3-2) was pasted at a position 22 mm from the bottom with the long side of the back sheet facing down.
The conjugate pad prepared in (3-1) was attached so that the distance between the right end of the conjugate pad and the left end of the reaction membrane was as shown in Table 1, respectively. In Comparative Example 3, the space between the conjugate pad and the reaction film was filled with an acrylic plate.
Next, the development pad which consists of a glass fiber pad (Millipore) cut into 17 mm x 6 mm was bonded together so that all might overlap with a reaction membrane 3 mm. Finally, a 10 mm × 6 mm sample addition pad and a 26.7 mm × 6 mm absorption pad (Nippon Pole) were stacked and pasted to prepare an immunochromatographic test device.

<< Example 2: Evaluation of immunochromatographic test device (1) >>
Using the immunochromatographic test device prepared above, the labeling substance remained on the conjugate pad and the presence or absence of background was confirmed.
Mycoplasma pneumoniae cultures were dissolved in the extract to prepare Mycoplasma pneumoniae solution-added dilutions at concentrations of 1 × 10 ⁵ and 1 × 10 ⁶ CFU / mL. The composition of the extract is 0.1 mmol / L phosphate buffer pH 7.5, 0.15 mmol / L NaCl, 1% Triton X-100, 1% BSA.

120 μL of the Mycoplasma pneumoniae solution-added diluted solution having the above concentration was dropped into each test immunochromatography kit, and the staining degree of the test line and the control line of the antibody-fixed membrane was determined 15 minutes later. The degree of staining was evaluated semi-quantitatively by comparing from the color sample for determination from “+” to “6+” from the weakest to the strongest dyeing. “-” Indicates a negative reaction.
At the same time, the presence or absence of the first labeled substance remaining on the conjugate pad was visually confirmed, and each was evaluated as “Yes” if a dye due to the labeled substance was observed, and “No” if no dye was observed. . Similarly, the presence or absence of background on the reaction film was visually confirmed, and when there was an increase in background, “Yes” was evaluated, and when there was no increase, “No” was evaluated.

Table 2 shows the results obtained using the 1 × 10 ⁶ CFU / mL Mycoplasma pneumoniae solution addition dilution. By arranging the development pad so as to form a space between the conjugate pad and the reaction membrane, the labeling substance remained on the conjugate pad and the background was not increased. The same tendency was obtained in the test using the 1 × 10 ⁶ CFU / mL Mycoplasma pneumoniae solution addition dilution.

<< Example 3: Evaluation of immunochromatographic test device (2) >>
An immunochromatographic test device was prepared in the same manner as in Example 1 except that the distance between the right end of the conjugate pad and the left end of the reaction membrane was changed, and labeling with the conjugate pad was performed in the same manner as in Example 2. The residue of the substance and the presence or absence of background were confirmed.

Table 3 shows the results obtained using the 1 × 10 ⁶ CFU / mL Mycoplasma pneumoniae solution addition dilution. In the test tool with a distance of 3 mm between the conjugate pad and the reaction membrane, the development pad and the back sheet are easy to contact, and in the test tool of 4 mm or more, the development pad is completely in contact with the back sheet. Not formed. The residual of the labeling substance and the background increase in the conjugate pad were not recognized as the distance between the conjugate pad and the reaction membrane was shorter. The same tendency was obtained in the test using the 1 × 10 ⁶ CFU / mL Mycoplasma pneumoniae solution addition dilution.

By using the immunochromatographic test device of the present invention, a highly accurate determination result can be obtained, which is industrially useful.
As mentioned above, although this invention was demonstrated along the specific aspect, the deformation | transformation and improvement obvious to those skilled in the art are included in the scope of the present invention.

1 ... Reaction membrane; 2 ... Conjugate pad; 3 ... Development pad;
4 ... Absorbing pad; 5 ... Back sheet; 6 ... Test line;
7 ... control line; 8 ... sample addition pad.

Claims (1)

A conjugate pad holding a labeling substance;
A reaction membrane disposed at a distance from the conjugate pad and having a site where a substance specific to the analyte is immobilized;
A deployment pad arranged to generate capillary action with both the conjugate pad and the reaction membrane, wherein the deployment is performed to form a space between the conjugate pad and the reaction membrane. An immunochromatographic test device, wherein a pad is arranged.

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